Amber is a natural, amorphous, polymeric glass, fossilized resin from plants having mechanical, dielectric, and thermal features common to other synthetic polymeric glasses, Poinar, G. O., Life in Amber, Stanford University Press, Stanford, Calif. (1992). The plant resins from which amber originates comprise complex mixtures of terpenoid compounds, acids, alcohols, and essential oils. As the resin ages, it becomes harder and forms a semifossilized product known as copal. Recently fossilized tree resins are considered copal (less than about 4 million years old), while older fossilized tree resins are generally considered amber. Amber and copal are better distinguished by studying various physical characteristics, such as melting point, hardness, solubility, and the like.
The oldest amber known was recovered from the Carboniferous Period of the Paleozoic era (360 to 286 million years old). Most of the well studied amber comes from the Cretaceous Period of the Mesozoic Era (65 to 144 million years ago) and the Tertiary Period of the Cenozoic Era (2 to 65 million years ago). For a comprehensive discussion of amber and its characteristics, see, Poinar, supra.
Amber appears to have a remarkable capacity to preserve biological materials, including cell organelles. For example, beginning in this century, scientists have observed the preservation of tissue in amber inclusions, see Poinar, supra at 266.varies.271. Specifically, well-preserved cells and cell components, including mitochondria and chromatin, have been observed in a 40 million year old fly fossilized in Baltic amber, Poinar and Hess, Science 215:1241-1242 (1982).
More recently, scientists have started to evaluate whether amber is capable of preserving intact ancient genetic material. Beginning in the mid-1980's, for example, scientists attempted to recover viable ancient biological materials, and specifically ancient DNA from a wide range of ancient sources. Early experiments to extract ancient DNA were conducted on museum skins, Higuchi et al., Nature 312:282-284 (1984), Thomas et al., J. Mol. Evol. 31:101-112 (1990), mummified tissues, Paabo, Nature 314:644-645 (1985) and Lawlor et al., Nature 349:785-788, (1991), bones, Hagelberg et al., Nature 342:485 (1989); Hagelberg et al., Phil. Trans. R. Soc. Lond. B. 333:399-407 (1991); Hanni et al., Acad. Sci. Paris Ser. III. 310:365-370 (1990); Hagelberg and Clegg, Proc. R. Soc. Lond. B. 244:45-50 (1991); Horai et al., Phil. Trans. R. Soc. Lond. B. 333:409-417 (1991); Hummel and Herrman, Naturwissenschaften 78:266-267 (1991), plant fossils, Golenberg, Nature 344:656-658 (1990), frozen woolly mammoths, Higuchi and Wilson, Federation Proc. 43:1557 (1984); Cherfas, Science 253:1354-1356 (1991), and ancient seeds, Rogers and Bendich, Plant Mol. Biol. 5:69-76 (1985), Rollo et al., Nature 335:774 (1988). However, these repeated attempts to isolate viable ancient DNA from fossilized materials entombed in amber were met with failure.
The first reported successful isolation of ancient DNA from amber fossils occurred in 1992, when DNA fragments were isolated from extinct bees, Proplebeia dominicana, preserved in 25-40 million year old Dominican amber, Cano et al., Med. Sci. Res. 20:249-251 and 619-622 (1992). Very recently, DNA from a 120-135 million year old weevil preserved in ancient Lebanese amber was PCR amplified and sequenced, Cano et al., Nature 363:536-538 (1993). These reports indicate that amber can, to some extent, preserve ancient genetic information.
Nonetheless, despite the reports of successful recovery of ancient DNA from amber and other ancient materials, considerable skepticism exists within the scientific community as to whether the genetic materials sequenced in these studies is truly ancient or attributable to modern-day contaminants. (See Ancient DNA, Springer-Verlag at 10, 62-64, 158-160, 221-222). Other scientists argue that due to the inherent instability of the DNA molecule, amber, it is impossible for viable DNA to remain intact for millions of years. Lindhal, Nature: 362:709-715 (1993).
Notwithstanding the wealth of work being conducted with respect to the extraction of ancient DNA from various sources, including amber, very little work has been conducted with respect to the recovery of ancient bacteria and other ancient biological materials preserved in amber or similar naturally-occurring resin materials. The meaning of this work further has been hindered by inconclusive results. For example, although bacterial rods and fungal spores (Micrococcus electroni, Bacillus electroni, Longibacillus electroni and Spirillum electroni), as well as pollen, were observed after dissolving amber in turpentine as early as 1929, Blunck, G., Bacterienneischlusse imm Berstein, Centraalblatt fur Mineralogie, Geologie und Palaontoligie (ABt. B, nomII 554-5) (1929), cited in, Poinar, G. O., Life in Amber at 350, Stanford University Press, Stanford, Calif. (1992). These organisms were attributed to modern-day laboratory contaminants. Id.
Similarly, reports of viable bacteria isolated from Paleozoic salts (Dombrosky. H. J., Zentr. Bakteriol. Parasitenk. Abt. I. 178:83-90 (1960); Dombrosky. H. J., Zentr. Bakteriol. Parasitenk. Abt. 1. 183:173-179 (1961)) have been attributed to modern bacterial contaminants, as pools of ancient microorganisms trapped in such soil sediments are likely to have become contaminated with more recent microorganisms via ground water infiltration. In 1983, intact bacterial cells were observed to be preserved in Mexican amber, Poinar, supra. Again, however, neither the age nor authenticity of the cells have been confirmed and contamination by modern bacteria is suspected. The results of each of these attempts to recover viable bacteria from amber thus have been inconclusive.
Intensive commercial development of microbes and microbial by-products for medical, industrial and agricultural applications and bioremediation have occurred during the last 80 years. Harvey, ed., Drugs From Natural Products; Pharmaceuticals and Agrochemicals, Ellis Horwood Ltd., England (1993) which is incorporated herein by reference. For example, the large scale scientific development of antibiotics was triggered in 1928 by work on Penicillin produced by the fungus Penicillium notatum. Likewise, intense use of microbial processes for industrial use increased in the 1940's with work on acetone using the anaerobic bacterium Clostridium acetobutylicum, Demain and Solomon, "Industrial microbiology and the advent of genetic engineering," Scientific American at pp. 3-11, W. H. Freeman and Co. San Francisco (1981). Relatively few species of microorganisms have presently been exploited for the production of antimicrobial compounds and other microbial by-products for use in medical or industrial processes. For example, only three groups of microorganisms (filamentous fungi, nonfilamentous bacteria and filamentous bacteria, or Actinomyces) are used to produce the bulk of antimicrobial compounds, and industrial microbial by-products.
In addition to producing antimicrobial compounds, microbes produce a wide variety of metabolites that have been used as other pharmaceutical applications including, but not limited to, cardiovascular and anti-inflammatory agents, immunoregulators, anti-tumor compounds, regulators of the central nervous system, and enzyme inhibitors. See e.g., Harwood, C. R., Biotechnology Handbooks: Bacillus at 294 et seq., Plenum Press, New York (1989). Microbial metabolites also serve as platform molecules for synthetic chemistry and rational drug design and are widely used as the basis of vaccine production. See, Harvey, ed., Drugs From Natural Products; Pharmaceuticals and Agrochemicals, Ellis Horwood Ltd., England (1993).
Microbes and microbial by-products have also been used in a variety of industrial processes, such as enzyme and vitamin production and fermentation. For example, B. subtilis produces a variety of thermostable serine protease enzymes that are widely used in laundry detergents. Id. Various species of fungi, including Saccharomyces, Aspergillus and Candida are used in the production of beer, wine, sake and other food products, as well as a variety of vitamins. Microbial by-products are also used in the production of cosmetics, biopolymers, surfactants, purine nucleosides, and phenolic germicides. Id.
More recently, due to increasing concern over environmental pollution, microbes and microbial by-products are being used for biopesticides and bioremediation. For example, B. sphaericus and B. thuringiensis are used commercially as biopesticides to combat a variety of plant and insect pests. See, Harwood at 309, supra. Similarly, species of bacillus, are routinely used in environmental clean-up of toxic pollutants. Id. at 313; Debabov, "The Industrial Use Of Bacilli" in The Molecular Biology Of The Bacilli, Vol. 1 (D.A. Dubnau, ed.), Academic Press, New York (1982) at 331-370, which is incorporated herein by reference.
Additionally, microbes and microbial by-products have been increasingly used in diagnostic assays. For example, B. subtilis is used in assays to detect streptomycin, penicillin and cathomycin. See, ATCC Catalog at 34 (1994) citing J. Bact. 45:408-409 (1943), J. Bact. 49:411 (1945), Appl. Microbiol. 4:307-310 (1956).
Changing patterns in animal and human health, agricultural, industrial and environmental processes demand a search for new sources of microbial by-products for medical, diagnostic and industrial applications. For example, numerous pathogenic microorganisms have become resistant to antibiotics currently derived from modern microorganisms, Cooksey, R. C., "Mechanisms of resistance to antimicrobial agents," Manual of Clinical Microbiology at 1099, 5th. Ed., Am . Soc. for Microbiology (1991). Similarly, many widely used agrochemicals have been shown to be toxic to flora and fauna and to cause contamination to water resources. Growing resistance to traditional antimicrobial compounds, new demands created by modern industrial processes, the need to develop alternatives to chemical pesticides and herbicides, and the need to eliminate environmental contaminants create opportunities for using novel microbes and their by-products. A comprehensive summary of the identity and existing uses of the microbial by-products known to date is set forth in Biotechnology, volumes 1-8, H. J. Rehm and G. Reed, editors, Verlag Chemie (1986), which is incorporated herein by reference.